ANXA1 and FPR signaling promotes immune tolerance by suppressing neutrophil extracellular traps after pediatric liver transplantation

Background: Immune tolerance after pediatric liver transplantation could reduce long-term immunosuppression, but biomarkers and mechanisms remain poorly defined. We investigated whether clinical multi-omics and experimental validation identify an innate immune program linked to tolerance, focusing on ANXA1/formyl peptide receptor (FPR) signaling and neutrophil extracellular traps (NETs).
Methods: Fifty-nine pediatric recipients who initiated immunosuppressant withdrawal were analyzed. Withdrawal outcomes were classified by graft function and biopsy findings. Liver biopsy scRNA-seq, baseline plasma proteomics, immunohistochemistry, multiplex immunofluorescence, and ex vivo primary neutrophil NET assays were integrated to define tolerance-associated signatures. Mechanistic validation used an allogeneic transplant model, the ANXA1 mimetic peptide Ac2-26, FPR antagonists, and neutrophil-like cells.

Results: Conventional clinical and biochemical variables did not reliably predict withdrawal outcome, whereas approximately 40 percent of recipients achieved immune tolerance and dysfunction in non-tolerant recipients reversed after treatment resumption. scRNA-seq showed that tolerance was not driven by selective expansion of T-cell subsets but by pathways related to innate immune communication and leukocyte responses. Baseline proteomics converged on neutrophil degranulation and NET-associated pathways. Clinical validation demonstrated higher graft ANXA1 expression and lower NET-forming capacity in tolerant recipients, while non-tolerant recipients showed a pre-activated neutrophil phenotype. In experimental models, Ac2-26 reduced neutrophil infiltration, CitH3 and MPO expression, and histologic injury; these effects were attenuated by FPR blockade, particularly FPR2 inhibition. ANXA1/FPR signaling also reduced PI3K/AKT/mTOR phosphorylation and shifted the immune milieu toward lower effector T-cell and higher Treg representation.

Conclusions: Immune tolerance after pediatric liver transplantation appears to emerge early at the level of innate immunity. Integrated clinical multi-omics and functional validation identify ANXA1/FPR-linked suppression of neutrophil activation and NET formation as a core tolerance-associated mechanism. This axis may provide clinically relevant biomarkers and a potential strategy for safer immunosuppression minimization.